Abstract

DNA double-strand breaks that initiate meiotic recombination are exonucleolytically processed. This 5′→3′ resection is a central, conserved feature of recombination but remains poorly understood. To address this lack, we mapped resection endpoints genome-wide at high resolution in Saccharomyces cerevisiae. Full-length resection requires Exo1 exonuclease and the DSB-responsive kinase Tel1, but not Sgs1 helicase. Tel1 also promotes efficient and timely resection initiation. Resection endpoints display pronounced heterogeneity between genomic loci that reflects a tendency for nucleosomes to block Exo1, yet Exo1 also appears to digest chromatin with high processivity and at rates similar to naked DNA in vitro. This paradox points to nucleosome destabilization or eviction as a defining feature of the meiotic resection landscape.

Although meiotic resection was first demonstrated experimentally ≥25 years ago and has long been known to be a fundamental step in recombination (10, 11), it remains poorly understood. In fact, detailed data are available only for one side of a single artificial DSB hotspot (4); whether this extrapolates to natural hotspots is unknown. We mapped resection endpoints genome-wide with high specificity, sensitivity, and spatial resolution. These maps answered long-standing questions about genetic control of resection, the handoff from MRX-Sae2 to Exo1, locus-to-locus variation in resection, resection kinetics, and interaction of resection machinery with chromatin.

Mapping DSB resection endpoints

We digested DSB 3′ tails with ssDNA-specific nucleases to generate sequencing libraries of ssDNA–dsDNA junctions that we compared with DSB maps from Spo11-oligo sequencing (Fig. 1, A and B, and table S1) (12). Two biological replicates were pooled for each strain or time point [Pearson’s correlation coefficient (r) = 0.95 to 0.99] (fig. S1, A and B).

Reads from resection endpoints should be meiosis-specific and Spo11-dependent and should flank DSB hotspots with defined polarity (fig. S1C). S1 sequencing (S1-seq) reads were hotspot-enriched with the expected polarity, and enrichment was absent from premeiotic samples and meiotic samples from the catalytically inactive spo11-Y135F mutant (Fig. 1, B and C, and fig. S1, D to F). S1-seq signal correlated well with Spo11 oligos (Fig. 1D), and reads spread further from hotspots over time in dmc1Δ (Fig. 1B and fig. S1, E, G, and H), reflecting known hyperresection (13). Thus, S1-seq is a sensitive and quantitative measure of DSB resection endpoints.

Resection endpoints extended ~200 to 2000 nucleotides (nt) from hotspot centers (mean 822 nt), with a positive skew (Fig. 1, C and E). This pattern resembles Southern blotting at HIS4LEU2 (range 350–1550, mean 800 nt) (Fig. 1E) (4), but S1-seq captured less abundant species at the distribution extremes. Genome-average profiles were smooth and left-right symmetric (Fig. 1C), but individual hotspots were heterogeneous, with peaks and valleys differing between hotspots or sides of the same hotspot (Fig. 1B and fig. S1E).

Tel1 promotes resection initiation and extension

Absence of Tel1 [orthologous to human ataxia telangiectasia mutated (ATM)] decreased resection length at HIS4LEU2 for early DSBs (15). This and other findings led to the proposal that Tel1 controls resection when DSB numbers are still low, whereas higher DSB numbers later allow Mec1 [ataxia telangiectasia and Rad3–related (ATR) in humans] to substitute (15). S1-seq data allowed us to test this model and uncovered that Tel1 acts at multiple steps.

S1-seq reads in tel1Δ fell closer to DSB hotspots at early (2 hours) and later (4 hours) time points, indicating shorter resection, but with peaks similar to those of wild type (Fig. 2, A and B). Although some tracts at 4 hours in tel1Δ matched the longest in wild type (Fig. 2B), DSBs remained hyporesected overall (Fig. 2, A and B). Therefore, Tel1 modulates resection length throughout meiosis, not just early.

Recombination intermediates

Accompanying resection signal, a weaker S1-seq signal with the “wrong” polarity was seen —for example, top-strand reads mapping left of hotspots (Fig. 1B and fig. S1, D and E). This signal was not resection from neighboring hotspots; it correlated with hotspot strength, was meiosis-specific and Spo11-dependent, and was essentially absent in dmc1Δ (Figs. 1, B and C, and 3A and figs. S1, E to H, and S3A). We conclude that this signal derives from S1-sensitive recombination intermediates (RIs), probably displacement (D) loops from strand exchange (fig. S3, B and C). Throughout this study, resection profiles were corrected by subtracting an estimate for the small amount of RI signal with the “correct” polarity that presumably lies under the resection signal (fig. S3, B and C, and supplementary text).

At 4 hours in wild type, RI reads had a positively skewed distribution, with a mean of 606 base pairs (bp) from hotspot centers, ~25% shorter than resection length (Fig. 3B). Wild type and sgs1 were indistinguishable (fig. S3D), but RI distances were shorter in tel1Δ consonant with modestly shorter resection tracts (Fig. 3B), suggesting that resection length influences RI position. In exo1-nd, RI distances were even shorter (Figs. 1F and 3B and fig. S3E). The similarity of RI distances (mean 350 bp) to resection lengths in exo1-nd suggests that much of the resection tract is used up making RIs, whereas RIs in wild type usually form within a DSB-proximal subregion of resection tracts.

We reasoned that quantitative differences between Spo11 oligos and S1-seq can reveal differences for life spans of recombination steps, because turnover of Spo11-oligo complexes is tied to meiotic prophase exit (17), whereas life spans of resection and RI signals reflect recombination progression more directly. Smaller chromosomes tend to incur more DSBs per kilobase because of negative feedback tied to engagement of homologous chromosomes (12, 17). It was proposed that smaller chromosomes take longer on average to engage homologous partners because multiple DSBs per chromosome are usually needed for successful engagement (17, 18). This hypothesis predicts that resected DSBs should persist longer on smaller chromosomes. Indeed, the ratio of S1-seq resection signal to Spo11-oligo counts at 4 hours correlated negatively with chromosome size; the yield of resected DSBs (S1-seq) per DSB formed (Spo11 oligos) was higher on smaller chromosomes (Fig. 3C). Anticorrelation was not seen at 2 hours (Fig. 3C), as expected because early DSBs have not had time to progress in recombination regardless of chromosome size. The ratio of RI signal to Spo11-oligo counts was uncorrelated with chromosome size (fig. S3F); thus, RI life span is governed by local features rather than chromosome-pairing kinetics.

The ratio of S1-seq resection reads to Spo11 oligos was lower for pericentromeric hotspots than for other subchromosomal domains (Fig. 3D). This apparently shorter life span could reflect delayed DSB formation and/or more rapid DSB repair, which is consistent with kinetochore components suppressing DSB formation and promoting sister chromatid recombination (19).

Modeling Exo1 mechanism and speed

To gain further insight into Exo1 resection, we tested a model in which Exo1 enters DNA at a Mre11-generated nick and digests DNA until dissociation. This model predicts that Exo1 run lengths follow a geometric distribution defined by the average probability of dissociation at each nucleotide step. We determined the geometric distribution providing the best fit to wild-type resection when combined with the distribution of presumptive Exo1 entry points (the endpoint distribution in exo1-nd) (Fig. 4A). This geometric model could be ruled out because it fit the data poorly, but shifting the geometric distribution provided a better fit (Fig. 4A). This alternative can be conceptualized in terms of a high probability of Exo1 resecting for a minimum distance (best-fit estimate of 220 nt), after which resection termination is geometrically distributed.

How fast is Exo1? In vegetative cells, unrepairable DSBs are continuously resected at ~4.4 kb/hour (8, 20, 21). Long-range resection by Exo1 only (in sgs1) is slower, ~1 kb/hour (8). To test whether the vegetative rate can explain meiotic resection, we performed Monte Carlo simulations to generate populations of resected DNA molecules to compare with S1-seq patterns (Fig. 4, B and C, fig. S4, and movies S1 and S2). Rates of 4 or 8 kb/hour were not fast enough; these speeds predicted that DSBs should be resected less than observed, particularly at early times. But simulations matched observations well when we used rates from 16 to 40 kb/hour, the latter being the value from single-molecule studies for Exo1 resecting naked DNA (22). Because rates of ≤8 kb/hour appear implausibly slow, we conclude that meiotic resection is faster than long-range resection in vegetative cells. Because ≥16 kb/hour is plausible, it suggests that Exo1 processes meiotic DSBs in vivo nearly as quickly as it degrades naked DNA in vitro. Hyperresection in dmc1Δ was slower, with an estimated average speed of 0.19 kb/hour (Fig. 4D). These findings illuminate differences between the extreme rapidity but limited length of wild-type meiotic resection, the slow continuity of hyperresection in the absence of Dmc1, and the moderate pace but unconstrained distance of long-range resection of unrepairable breaks in vegetative cells.

Chromatin shapes the resection landscape

Verifying that strong peaks in S1-seq resection profiles were not a sequencing artifact, S1 treatment converted resected DSB fragments at the GAT1 hotspot from the usual featureless smears into discrete banding patterns on Southern blots (Fig. 5A and fig. S5A). Prominent bands occupied similar positions in mutants with different average resection lengths, so banding is a reproducible feature at GAT1. In contrast, S1 generated no discrete banding at HIS4LEU2 (fig. S5B), explaining why preferred resection endpoints were not previously uncovered (4). Additionally, the spatial resolution of resection analyses in vegetative cells would have been inadequate to detect such heterogeneity if it were present.

Stereotyped nucleosome positions around hotspots allowed us to test the hypothesis that this heterogeneity reflects chromatin structure. Most yeast promoters have a nucleosome depleted region (NDR), where many DSBs form, flanked by positioned nucleosomes with the transcription start site (TSS) in the first (+1) nucleosome (Fig. 5B) (12, 23). When S1-seq data from wild type were averaged around +3 nucleosomes, the broad peak showed modest scalloping in register with average nucleosome occupancy (Fig. 5C). Speculating that variation in nucleosome positions blurs the chromatin signature, we compiled S1-seq averages for three groups of genes divided by linker width between +3 and +4 nucleosomes (Fig. 5D). An S1-seq peak overlapping left edges of +4 nucleosomes moved progressively rightward with increasing linker width (Fig. 5D, arrows). The widest group also accumulated reads within linkers. Similar patterns for the shorter resection tracts in tel1Δ were seen for +2 and +3 nucleosomes (fig. S5, C and D), and +1 and +2 nucleosomes yielded similar patterns for Mre11-dependent clipping in exo1-nd (Fig. 5, E and F). These findings establish a spatial correlation between nucleosomes and preferred endpoints for both the Mre11 and Exo1 resection steps. Patterns for Exo1 fit with digestion proceeding partway into nucleosomes, with increasing likelihood of resection termination as Exo1 approaches the nucleosome dyad, possibly reflecting greater stability of histone-DNA binding near the dyad (24). RIs tended to peak within nucleosome linkers (fig. S5E), indicating that RIs are also influenced by chromatin, possibly the recombination partner’s.

Conclusions

Given chromatin structure at hotspots, Mre11 incision often occurs in +1 or +2 nucleosomes, and Exo1 then traverses several nucleosomes’ worth of DNA (Fig. 5B). This creates an apparent paradox: Feeble activity of Exo1 on nucleosomal substrates in vitro (26) and the tendency for resection to stop near nucleosome boundaries in vivo indicate that nucleosomes are a potent block to Exo1. Yet, Exo1 resects several hundred nucleotides quickly and with high apparent processivity in vivo, as though nucleosomes are initially little or no barrier at all.

To resolve this paradox, we propose that nucleosomes are destabilized or removed before digestion by Exo1, with a constraint on resection length being how many nucleosomes are removed (fig. S5G). Additional factors may include inhibitors of Exo1, spatially regulated Exo1 activity, and/or iterative Exo1 loading (fig. S5H). Nucleosome eviction during resection has been proposed for vegetative yeast and somatic mammalian cells, but experimental support is indirect and conflicting and has been unable to distinguish whether chromatin changes are a prerequisite for or consequence of resection (27–29). Our parameterization of resection speed, apparent processivity, and preferred endpoint positions clearly favor this model. Chromatin immunoprecipitation also supports nucleosome disassembly near unresected DSBs being repaired by nonhomologous end-joining in human cells (29). Chromatin remodelers implicated in vegetative resection (30) are candidates for nucleosome destabilization in meiosis. DSB resection in mouse meiosis extends a similar average distance as in yeast and is inferred to traverse multiple nucleosomes (31). Conservation of the scale of DSB-provoked chromatin remodeling would parsimoniously unify resection mechanisms in these distant species.

If nucleosomes are evicted, we infer that this occurs after DSB formation because Spo11 rarely cuts within nucleosomes (12). Furthermore, the chromatin signature in exo1-nd implies that full eviction occurs after Mre11-dependent incision positions are established. Unresected DSBs in wild type may indicate that Mre11 incision is rate-limiting, as in Schizosaccharomyces pombe (32). Tel1 may promote Mre11-dependent incision given that there are more unresected DSBs in its absence. Tel1 also regulates resection distance without changing preferred endpoints. A minor role of Tel1 in mitotic DSB resection has been reported (33), and ATM is proposed to destabilize chromatin during nonhomologous end-joining in human cells (29). We speculate that Tel1 controls efficiency or distance over which nucleosome destabilization occurs via effects on chromatin remodelers, histone modifications, or both. S1-seq gives a selective, sensitive, and quantitative measure of DSB resection tracts and should be applicable to other settings and organisms.

Acknowledgments: We thank A. Viale (MSKCC Integrated Genomics Operation) for sequencing and N. Socci (MSKCC Bioinformatics Core) for mapping S1-seq reads. Core facilities were supported by NIH grant P30 CA008748. We thank M. Neale for discussions, N. Hunter and R. Liskay for strains, and S. Tischfield for discussions and help with base composition analysis. This work was supported by NIH grants R01 GM058673 and R35 GM118092. E.P.M. was supported by a Helen Hay Whitney Foundation Fellowship, and S.Y. was supported by a Kuro Murase MD-JMSA Scholarship. Sequencing data are at the Gene Expression Omnibus (accession GSE85253). The authors declare no competing financial interests. Correspondence and requests for materials should be addressed to S.K. (s-keeney@ski.mskcc.org). E.P.M. developed S1-seq and performed experiments. S.Y. developed in silico modeling of resection length and speed. E.P.M. and S.K. designed the study, analyzed data, and wrote the paper, with contributions from S.Y.